Dietary supplements containing natural ingredients

ABSTRACT

The invention provides a dietary supplement comprising at least one flavonoid source and an enzyme, that is effective for inhibiting in vivo platelet activity and LDL cholesterol oxidation in a mammal at a dosage of about 30 mg/Kg or less. The supplement may contain flavonoid sources found in grape seed extracts, grape skin extracts, bilberry extracts, ginkgo biloba extracts or the flavonoid quercetin. The supplement may also contain fungal proteases, acid stable proteases and bromelain. The invention further provides a method for using the dietary supplement and an article of manufacture containing the supplement.

RELATED APPLICATIONS

This application is a U.S. National Phase application of PCT PatentApplication No. PCT/US98/16181, filed Aug. 5, 1998, which is acontinuation of U.S. patent application Ser. No. 08/907,317, filed Aug.6, 1997, now abandoned.

BACKGROUND

1. Technical Field

The invention relates to dietary supplements containing naturalingredients.

2. Background Information

Coronary artery disease, myocardial infarction, stroke, and othervascular occlusions are major health concerns. A common characteristicof these diseases is the atherosclerotic process, or the narrowing ofarteries. Blood platelets contribute to the development and progressionof the atherosclerotic process by releasing growth factors, chemotacticsubstances and other factors that accelerate the atheroscleroticprocess. In addition, platelet aggregation at or near the point ofarterial damage contributes to the development of atherosclerosis andacute platelet thrombus formation.

Low density lipoprotein (LDL) cholesterol is also associated withatherosclerosis. It has been proposed that nonatherogenic LDLcholesterol circulating in the blood is converted to atherogenic LDLcholesterol through oxidation of polyunsaturated lipids, which leads tomodification of the apoprotein.

Physicians use various drugs, such as aspirin, to treat atheroscleroticconditions. Aspirin, however, is not without negative side effectsincluding gastrointestinal irritation. Interventions such as angioplastyare also available to dilate stenosed arteries thereby increasing bloodflow. Interventional techniques, however, produce intimal and medialartery damage and expose thrombogenic surfaces. As such, restenosis andthe incidence of sudden coronary death following angioplasty is a majorconcern for patients with known or suspected coronary artery disease.

Given the grave consequences of atherosclerosis and the costs associatedwith medical treatments, there is a need for pharmacologic andnutritional interventions that are useful for preventing the occurrenceand reoccurrence of these conditions.

Epidemiological studies have noted an inverse correlation between theintake of dietary flavonoids from fruits and vegetables and death fromcoronary artery disease. This correlation is thought to arise from theantioxidant and platelet inhibition properties of flavonoids found infruits and vegetables.

Certain flavonoids, including those found in grape seed and grape skinextracts, have been associated with the beneficial health effectsobserved for aspirin, but without the negative side effects attributedto aspirin. Nevertheless, flavonoid bioavailability or activity is lowin many sources of flavonoids. As such, certain dietary sources offlavonoids require large doses to be useful. As a result, many sourcesof flavonoids are impractical, too costly, or both to be useful on adaily basis.

SUMMARY

The present invention involves the discovery that the combination ofcertain flavonoids and enzymes in the form of a dietary supplementreduces the dosage of supplement needed to effectively reduce plateletactivity and LDL cholesterol oxidation in a mammal. The presentinvention further involves a method to treat conditions associated withplatelet activity and LDL cholesterol oxidation by administeringcombinations of flavonoids and enzymes to reduce platelet activity andLDL cholesterol oxidation.

In one aspect, the invention features a dietary supplement containing atleast one flavonoid source and an enzyme wherein the supplement iseffective for inhibiting platelet activity and LDL cholesterol oxidationin a mammal at a dosage of about 30 mg/Kg or less. One example of adietary supplement in accordance with the invention is PROVEXCV™. It isto be understood that a dosage of supplement as used herein refers tothe combined weight of the flavonoid source or sources and enzyme.Further, other ingredients such as fillers, lubricants, carriers and thelike may be included as additional ingredients.

The flavonoid source or sources in the dietary supplement may be derivedfrom grape seed extracts, grape skin extracts, ginkgo biloba extracts,bilberry extracts or quercetin. The enzymes may include fungalproteases, acid stable proteases, neutral stable proteases, alkalinestable proteases or bromelain.

In another aspect, the invention features a dietary supplementcontaining at least one flavonoid source wherein the supplement iseffective for inhibiting platelet activity and LDL cholesterol oxidationin a mammal at a dosage of about 30 mg/Kg or less. Preferably, a dietarysupplement in accordance with this aspect further includes an enzymethat substantially reduces the dosage required to achieve suchinhibition.

In another aspect, the invention features a method to inhibit plateletactivity or LDL cholesterol oxidation or both in a mammal byadministering a dietary supplement containing a flavonoid source and anenzyme wherein the supplement is effective for reducing plateletactivity or LDL cholesterol oxidation at a dosage of about 30 mg/Kg orless. The method may also be used to treat a condition that isassociated with platelet activity or LDL cholesterol oxidation byadministering a dietary supplement containing a flavonoid source and anenzyme that are effective for reducing platelet activity or LDLcholesterol oxidation.

In another aspect, the invention features an article of manufacturecontaining a dietary supplement that is effective for reducing plateletactivity and LDL cholesterol oxidation contained within a packagingmaterial wherein the packaging material is labeled to indicate that thedietary supplement is effective for reducing platelet activity or LDLcholesterol oxidation or both in a mammal at a dosage of about 30 mg/Kgor less. In another embodiment of the article of manufacture, thepackaging material may be labeled to indicate that the dietarysupplement is useful to treat a condition that is associated withplatelet activity or LDL cholesterol oxidation.

Unless otherwise defined, all technical and scientific terms andabbreviations used herein have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention pertains.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. Otherfeatures and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic diagram describing the animal model forproducing stenosed arteries to measure changes in coronary blood flowthat are observed when periodic thrombosis occurs in order to mimic theproblems that occur in patients with narrowed coronary arteries due toatherosclerosis.

FIG. 2 is a strip chart recording showing the repeated platelet-mediatedthrombosis formation followed by embolization, which produces thecyclical flow reductions (CFR's) that are observed for blood flowmeasured in stenosed arteries produced by the Folts model.

FIG. 3 is a strip chart recording showing the elimination of CFR'sfollowing ingestion of PROVEXCV™ due to the in vivo platelet inhibitingproperties of PROVEXCV™, which is a preferred dietary supplement inaccordance with the invention.

FIG. 4 is a strip chart recording showing that the CFR's eliminated byadministering PROVEXCV™ did not reappear when epinephrine (adrenaline)was administered two and one half hours after administering PROVEXCV™.

FIG. 5 shows a schematic diagram describing the whole blood aggregometrymethod used herein to measure blood platelet activity ex vivo.

FIG. 6 is a graph showing the time course of LDL cholesterol oxidationmonitored at 234 nm in the presence of vitamin E, ProVex Plus™ orPROVEXCV™.

FIG. 7 shows a graph of the level of protection against LDL cholesteroloxidation provided by PROVEXCV™.

FIG. 8 is a graph depicting the rate of LDL cholesterol oxidation.

DETAILED DESCRIPTION

The present invention involves the discovery that the combination ofcertain flavonoids and enzymes in the form of a dietary supplement willreduce the dosage of supplement needed to effectively reduce plateletactivity and LDL cholesterol oxidation in a mammal. The presentinvention further involves a method to treat conditions associated withplatelet activity and LDL cholesterol oxidation by administeringcombinations of flavonoids, extracts and enzymes to reduce plateletactivity and LDL cholesterol oxidation.

Coronary artery disease, cerebrovascular disease and peripheral vascularocclusions are characterized by arterial narrowing. If arterialnarrowing is further compromised by thrombotic occlusions, blood flow isfurther decreased and myocardial infarction or stroke may occur. Theseconditions are associated with platelet activity and LDL cholesteroloxidation. Current treatments for these types of vascular occlusions,such as angioplasty, result in intimal and medial damage to arteries,which can result in restenosis, acute thrombosis, myocardial infarctionand sudden coronary death in patients. Moreover, platelet-mediatedthrombus formation also plays a role in unstable angina, myocardialinfarction and restenosis following angioplasty or atherectomy. Thepresent invention provides a dietary supplement and a method to preventnew and recurrent thrombotic occurrences by administering a dietarysupplement that inhibits platelet activity and LDL cholesteroloxidation.

Methods to Evaluate Platelet Activity

1. The Folts Model

The Folts coronary thrombosis model (“Folts model”) is used to studyplatelet aggregation and thrombosis in a variety of animal models.Folts, J. D., Circulation (Supplement 4), 83:3-14 (1991); Folts, J. D.,J. Cardiovasc. Drugs & Therapy, 9:31-43 (1995); Folts et al.,Circulation, 54:365-370 (1976). The Folts model mimics the problems thatoccur in patients with narrowed coronary arteries due toatherosclerosis.

To use the Folts model, the chest of a fully anesthetized animal isopened and the heart exposed. Although the experiments described hereininvolved dogs, other animals amenable to the Folts model include pigs,rabbits, monkeys and other similar animals. Moreover, the Folts model isa model for the clinical syndrome of unstable angina and otherplatelet-mediated thrombotic events in humans. Samama et al., Thromb.Haemost., 68:500-05 (1992); Raeder et al., Am. Heart J., 104:249-253(1983); Sherry, S., Cardiovasc. Rev. Rep., 5:1208-19 (1984); Ikeda etal., J. Am. Coll. Cardiol., 21:1008-1017 (1993).

As is shown in FIG. 1, the circumflex coronary artery is dissected outand a coronary artery flow probe is placed on the artery. The coronaryartery flow probe continuously measures blood flow through the arterywithout interfering with the artery. The artery is then clamped tocreate intimal and medial damage to the coronary artery. A plasticconstricting cylinder is placed around the outside of the coronaryartery at the point of arterial damage to reduce the internal diameterof the coronary artery. The extent of stenosis or diameter reductionproduced by the cylinder is altered using an angioplasty balloon orother suitable method.

The coronary artery flow probe measures changes in the coronary arteryblood flow when clots periodically develop. When an artery is stenosed,platelets periodically aggregate in the narrowed and damaged part of thecoronary artery. Platelet aggregation produces a thrombosis (blood clot)that increases in size, and gradually cuts off the coronary artery bloodflow. As the artery becomes occluded, pressure builds up behind theplatelet aggregation and may dislodge the clot forcing it through thestenosis, which causes a sudden restoration of coronary blood flow inthe narrowed artery. Platelet aggregation in a damaged artery that issufficient to decrease blood flow is referred to as acute plateletthrombus formation (APTF).

Thrombotic arterial occlusion in human atherosclerotic arteries isthought to begin with the exposure of the thrombogenic surface beneaththe intimal lining of an artery. In addition, current informationsuggests that a variety of different stimuli recruit platelets to adhereand aggregate at an exposed thrombogenic surface. It is to beunderstood, however, that the scope of the present invention is notlimited by a particular theory of pathogenesis.

Repeated platelet-mediated thrombosis formation followed by embolizationand the observed concomitant changes in blood flow are referred to ascyclical flow reductions (CFR's) or cyclical flow variations (CFV's).This document will use the term CFR's. CFR's have also been observed indamaged and narrowed arteries in humans with femoral artery or coronaryartery disease. Folts et al., Tex. Heart Int. J., 9:19-27 (1982);Eichorn et al., J Am. Coll. Cardiol., 17:43-52 (1991). As FIG. 2 shows,CFR's are detected and monitored by measuring coronary artery bloodflow. Although FIG. 2 is illustrative of CFR's observed for coronaryblood flow, it is to be understood that the Folts method is applicableto arterial flow in general (e.g., femoral arterial flow). In addition,techniques needed to use the Folts model and variations of the model aredescribed in Folts, J. D., Circulation (Supplement 4), 83:3-14 (1991);Folts, J. D., J. Cardiovasc. Drugs & Therapy, 9:31-43 (1995) and thereferences therein.

The frequency or number of CFR's per unit of time is a direct measure ofin vivo platelet activity. Folts, J. D., J. Cardiovasc. Drugs & Therapy,9:31-43 (1995). By measuring the frequency and extent of CFR's instenosed arteries, the Folts model can be used to evaluate agents thatwill alter platelet activity. Folts, J. D., J. Cardiovasc. Drugs Ther.,9:31-43 (1995). As such, the Folts model identifies platelet inhibitors,the extent of inhibitor activity, the effective dosages of inhibitors,the duration of inhibition and the ability of an inhibitor to counteractplatelet agonists.

Aspirin provides an illustrative example of the Folts model. The Foltsmodel demonstrated that aspirin inhibits platelet activity andeliminates CFR's under the experimental conditions. Folts et al., Clin.Res., 22:595 (1974) (abstract); Folts, J., Circulation (Supplement 4),83:3-14 (1991); Folts, J. D., J. Cardiovasc. Drugs Ther., 9:31-43(1995). Aspirin is known to inhibit platelet activity at a dosage of 5mg/Kg. When platelet activity is inhibited with aspirin under conditionsused in the Folts model, platelet activity can be renewed by increasingthe concentration of epinephrine (adrenaline) or norepinephrine in theblood. This increase in adrenaline occurs naturally in many people whenthey are stressed, scared, anxious or exercising. Folts, J. D. & Rowe,G. G., Thromb. Res., 50:507-516 (1988). In addition, increasing thearterial stenosis will also cause a recurrence of CFR's abolished withaspirin. Folts, J., Circulation (Supplement 4), 83:3-14 (1991). As such,the Folts model provides an effective tool to evaluate inhibition ofplatelet activity.

2. The Platelet Aggregometry Test

A second method to measure platelet activity in humans or animals is thewhole blood platelet aggregometry test. Equipment and methods to performwhole blood platelet aggregometry evaluations described and used hereinare available from Chronolog Inc., 2 West Park Rd., Haverton, Pa. 19083.To perform the test, a blood sample is drawn by standard methods, whichis then placed in a plastic tube. A pair of electrodes is placed in theblood sample to measure the electrical resistance of the blood sample.The electrical resistance of blood is normally low due to the numerousions and electrolytes found in blood. A known platelet aggregationstimulus, such as adenosine diphosphate (ADP) or collagen, is then addedto the blood sample to activate the platelets. Activated platelets willadhere to the electrodes used in a platelet aggregometry test. As shownin FIG. 5 (right side), the electrical resistance of the blood sampleusually increases in a sigmoidal fashion as platelets aggregate on theelectrodes. If a subject is administered a substance that inhibitsplatelet activity, the increase in resistance following the addition ofa platelet stimulus will be smaller.

Aspirin provides an illustrative example of the use of the bloodaggregometry test. A blood sample taken from a human will have abaseline level of platelet activity. Addition of ADP increases theresistance of the blood sample. See FIG. 5, right side, “ADP” curve. Asecond blood sample is withdrawn two hours after the person is given a325 mg tablet of aspirin. Platelet activation following the addition ofADP does not increase platelet activity to the level observed for theinitial blood sample. See FIG. 5, right side, “ADP+ASA” curve. Thedecrease in blood resistance observed is expressed as a percentagedecrease in ex vivo platelet activity caused by aspirin. Whenepinephrine levels of a person, who has ingested aspirin, are raisedprior to withdrawing a blood sample, the inhibitory effects observed foraspirin are diminished. See FIG. 5, right side, “ADP+ASA+EPI” curve.Folts, J. D., J. Cardiovascular Drugs& Therapy, 9:31-43 (1995);Ingerman-Wojenski & Silver, Thromb. Haemost., 51:154-156 (1984).

Aspirin also responds similarly when collagen is used to stimulateplatelet activity. When collagen is used to stimulate platelet activityin the blood aggregometry test, aspirin decreases platelet activity byabout 30-40%. Therefore, the blood aggregometry technique may also beused as another measure of platelet inhibitory effectiveness produced bydrugs or flavonoids.

3. Evaluating LDL Cholesterol Oxidation

LDL cholesterol oxidation or protection therefrom can be determined byseveral methods. Kleinveld et al., Clin. Chem., 38(10):2066-2072 (1992)describes the methods for the LDL oxidation experiments described andused herein. A preferred way to practice the method encompassesextracting LDL cholesterol from whole blood samples collected from asubject using standard techniques. The extracted LDL cholesterol isdivided into control and experimental samples. LDL oxidation iscatalyzed by adding a copper ion solution to each LDL cholesterolsample. Copper ions are known to catalyze and enhance LDL cholesteroloxidation.

Control LDL cholesterol samples contain extracted LDL cholesterol andcopper ions. The control samples provide a baseline measure of theantioxidants in the diet of the subject from whom the blood was drawnand in the LDL cholesterol prepared therefrom. Test LDL cholesterolsamples are combined with copper ions in the presence or absence ofadditional compounds such as dietary supplements that may act asantioxidants.

Additionally, a subject may be administered an antioxidant by standardtechniques prior to collecting a blood sample, which facilitates themeasurement of antioxidant properties in vivo. To measure in vivo LDLcholesterol antioxidant properties, whole blood samples are obtainedbefore and after administering an antioxidant source to be tested.

Copper ions accelerate the production of conjugated dienes in LDLcholesterol oxidation, which absorb light at 234 nm. Therefore, LDLcholesterol oxidation is monitored by following the increase inabsorbance at 234 nm as a function of time as shown in FIG. 6.Antioxidants prolong the onset of diene production in LDL cholesterol.As a result, LDL cholesterol samples containing antioxidants can becompared by computing the time elapsed before LDL cholesterol oxidationis observed (lag time). See FIG. 7. The lag time is the best indicatorof the LDL cholesterol protection from oxidative damage under the LDLoxidation experimental conditions described herein. Longer lag timesdenote better antioxidant properties.

Inhibiting Platelet Activity and Decreasing LDL Cholesterol Oxidation

Many complex factors lead to atherosclerosis, coronary artery diseaseand related conditions. Among these factors, it is known that plateletinteraction with arterial walls can enhance the atherosclerotic process.Furthermore, increased platelet activity is intimately involved in boththe development of atherosclerosis and the transient and permanentoccurrence of thrombotic events. Therefore, factors that reduce plateletactivity on a daily basis should inhibit the development or occurrenceof atherosclerosis, coronary artery disease and related conditions.Folts et al., J. Myocard. Ischemia, 6(8):33-40 (1994).

Epidemiological studies reveal a inverse correlation between theconsumption of foods high in antioxidant flavonoids or vitamin E withreduced coronary heart disease. Hertog et al., Lancet, 342(8878):1007-11(1993); Waterhouse et al., Hypernutritious Foods, Agscience, Inc.,Auburndale, Fla., 219-238 (1997). Food sources known to containflavonoids include red wine, beer, grape juice, fruits and vegetables.Extracts from grape seeds, grape skins, ginkgo biloba, bilberry andother similar fruits, vegetables and herbs are also flavonoid andantioxidant sources.

There are, however, hurdles that must be overcome to effectively obtainthe benefits that are associated with flavonoids and antioxidants. Onehurdle is the bioavailability of the active ingredients in dietarysources of flavonoids. When bioavailability or activity is low, a personmust consume large quantities of a platelet inhibitor or antioxidantdietary supplement to receive the benefits associated with a plateletinhibitor or antioxidant. As a result, low bioavailability may render adietary supplement impractical, too costly or both for many consumers.

In addition, the effectiveness of the platelet inhibition andantioxidant characteristics associated with individual flavonoids variesbetween particular flavonoids, e.g., flavonoids from grapes are betterplatelet inhibitors in human volunteers and animals than flavonoidsfound in citrus fruit juices. Folts et al., J. Am. Coll. Cardioloqy,29(2)(Supplement A):303A (1997); Folts, J. D., J. Am. Coll. Cardiology,29(2)(Supplement A):226A (1997); Folts et al., J. Am. Coll. Cardiology,29(2)(Supplement A):180A (1997).

Moreover, the environmental conditions within the blood stream are notstatic. To be effective, a dietary supplement must be effective under avariety of conditions including various degrees of stress, excitementand exercise, which tend to elevate platelet activity levels.

Finally, many dietary sources of flavonoids also contain unwantedconstituents such as the alcohol in wines and beers and the sugars andcalories in grape juice. There are also toxicity concerns for variousflavonoids and drugs. For example, aspirin has well documented sideeffects that include gastrointestinal irritation, loss of effectivenessin the presence of epinephrine and the inability to inhibit CFR's underextreme stenosis circumstances.

Therefore, it was hypothesized that it would be useful to create adietary supplement that would inhibit platelet activity and LDLcholesterol oxidation under a variety of conditions in a practical andcost effective manner. It was further hypothesized that a key feature ofa useful dietary supplement would be to increase the bioavailability ofa dietary supplement by including constituents to enhance the absorptionof the dietary supplement active ingredients. The present inventor hasdiscovered such a supplement and the supplement is described and claimedherein.

In a first embodiment, the invention provides a dietary supplementcontaining at least one flavonoid source and an enzyme that areeffective for inhibiting blood platelet activity and LDL cholesteroloxidation in a mammal at a dosage of about 30 mg/Kg or less. The dietarysupplement may further contain flavonoid sources such as grape seedextracts, grape skin extracts, ginkgo biloba extracts, quercetin,bilberry extracts or any other specific flavonoid. The “flavonoidsource” may be derived from any source and may include synthetic orpurified flavonoids from known sources. The flavonoid source may alsobe, for example, one or more flavonoids determined to be highly activealone or as a combination wherein the flavonoid is isolated from acomplex mixture, such as a plant extract, containing numerousflavonoids.

While not bound by a particular theory of action, it is believed thatthe constituents of the presently disclosed dietary supplement actsynergistically by increasing bioavailability or pharmacologicinteractions, to inhibit blood platelet activity and inhibit LDLcholesterol oxidation. In a related aspect of the invention, the dietarysupplement remains effective for reducing platelet activity andinhibiting LDL cholesterol oxidation in the presence of elevatedplatelet agonist (e.g., epinephrine) concentrations.

Examples of dietary supplement formulations may include one or more ofthe following constituents: fruit extracts, vegetable extracts,digestive enzymes, herbs, flavonoids, antioxidants and other similaritems.

Illustrative examples of useful digestive enzymes include pepsin,papain, fungal proteases, acid stable proteases, neutral stableproteases, alkaline stable proteases and bromelain.

Illustrative examples of useful flavonoids include catechins,procyanidins, proanthocyanidins, quercetin, rutin and glycosidic formsof the flavonoids.

Dietary supplements of the present invention may be delivered orally,intravenously, subcutaneously, sublingually, intragastricly, as aphytosome or by any acceptable delivery method. In addition, the dietarysupplement may be combined with any suitable carrier to facilitatedelivery. Dietary supplements tested herein using the Folts model wereadministered either intravenously or intragastricly. Absorption throughthe stomach lining usually takes 2-4 hours. As such, dietary supplementsadministered intragastricly may not affect CFR's until about 2-4 hoursafter administering a dietary supplement.

Commercial dietary supplements are generally formulated to be givenorally. Useful forms of administration for dietary supplements includepills, pastes, powders, liquids and other common oral formulations.Preparations of the dietary supplements may be manufactured using commonmanufacturing techniques for the various forms of the supplementdescribed herein. The dietary supplements described herein additionallymay contain magnesium stearate as a lubricant to facilitate the deliverythe supplement into capsules. Magnesium stearate is generally used atconcentrations from 1-4 mg/capsule, preferably 1-2 mg/capsule.

The Folts model may be used to identify dietary supplement formulationsthat decrease platelet activity. A dog coronary artery is prepared toexhibit CFR's as described herein. The dog is then administered measureddoses of a dietary supplement by one of the approved methods. The effectthat the dietary supplement dose has on the observed CFR's is monitored.By using the Folts model, the effective dose, half life and extent ofabsorption can be determined. Blood samples taken from the dog provideinformation concerning supplement concentrations and platelet activitylevels within the blood stream.

Additional experiments may be used to evaluate the characteristics ofuseful dietary supplements. For example, the stenosis severity can beincreased following administering a dietary supplement to determine ifCFR's reoccur. In addition, platelet agonists administered at varioustime points preceding or following administering a dietary supplementwill identify dietary supplements that can sustain CFR elimination underplatelet activating conditions. Useful agonists include epinephrine andnorepinephrine. Useful doses of epinephrine for this purpose includeepinephrine administered intravenously at 0.2 μg/Kg/min for 15 minutes.

Human volunteers or patients with human coronary artery disease may alsobe administered the dietary supplements described and claimed herein andvariations thereof to evaluate the efficacy of the dietary supplementusing platelet aggregometry and LDL cholesterol oxidation experiments asdescribed herein.

Useful dietary supplement formulations may include one or more of thefollowing: grape seed extract, grape skin extract, ginkgo bilobaextract, bilberry extract, quercetin and an enzyme blend. The dietarysupplement may be formulated by weight in the following manner: grapeseed extract at 12% w/w, grape skin extract at 20% w/w, ginkgo bilobaextract at 10% w/w, bilberry extract at 10% w/w, quercetin at 24% w/wand an enzyme blend at 24% w/w.

As such, a useful dietary supplement containing flavonoids and enzymesin accordance with the present invention is PROVEXCV™. PROVEXCV™ isavailable from Melaleuca, Inc., Idaho Falls, Id.

PROVEXCV™ contains the following ingredients per 380 milligram capsule:

Ingredient % Formulation Amount Grape Seed Extract  12%  45 mg GinkgoBiloba Extract  10%  38 mg Bilberry Extract  10%  38 mg Grape SkinExtract  20%  76 mg Quercetin  24%  92 mg Enzyme Blend  24%  91 mg Total100% 380 mg

A supplement may also be prepared, in accordance with the presentinvention, to include all of the above ingredients except the EnzymeBlend.

The enzyme blend used in each 380 milligram capsule of PROVEXCV™contains the following enzymes:

Ingredient % of Enzyme blend Activity Units Fungal Protease 20053  25%11,250 HUT Fungal Protease 20054  25% 11,250 HUT Acid Stable Protease 25% 3,375 SAPU Bromelain  25% 5,760,000 PU Total 100%

The fungal proteases 20053 and 20054 are enzyme mixtures of acid,neutral and alkaline protease enzymes. HUT activity is the activity ofan enzyme measured in the FCC HUT assay, which is based on thehydrolysis of denatured hemoglobin. One HUT unit is defined as thatamount of enzyme that produces a hydrosylate whose absorbance at 275 nmis equal to a solution of 1.10 mg/ml of tyrosine in 0.006 N HCl in oneminute. SAPU activity is measured in the FCC SAPU assay, which is basedon hydrolysis of Hammarstan casein substrate. One SAPU unit is definedas that amount of enzyme that will liberate one μmole of tyrosine perminute at pH 3 and 37° C. PU activity is the activity of an enzymemeasure in the FCC PU assay, which is based on the hydrolysis of casein.One PU unit is defined as the amount of enzyme that liberates 1 μg oftyrosine per hour at pH 6.0 and 40° C. More information concerning theabove referenced enzymes is available from the National Enzyme Company,Forsyth, Mo., (417)-546-4796 through technical bulletins concerning theenzymes.

The ingredients contained in PROVEXCV™ can be obtained from thefollowing sources. The actual sources used are indicated by underliningthe supplier.

Ingredient/Extract Available from Grape Seed Extract Indena - Milan,Italy Polyphenolics - Canandaigua, NY InterHealth - Concord, CA Tri-KIndustries-Fanerson, NJ Ginkgo Biloba Extract Indena - Milan, ItalyWeinstein Nutritional - Irvine, CA OptiPure - Los Angles, CA BotanicalsInternational - Long Beach, CA Bilberry Extract Indena - Milan, ItalyOptiPure - Los Angeles, CA Chemco Industries - Los Angeles, CA GrapeSkin Extract Freeman Industries - Tuckahoe. NY Weinstein Nutritional -Irvine, CA Brucia Extracts - California Quercetin WeinsteinNutritional - Irvine, CA Botanicals International - Long Beach, CATriarco Industries - Wayne, NY Enzyme Blend National Enzyme Co. -Forsyth. MO MakWood - Thiensville, WI Botanicals International - LongBeach, CA Citrus Extract Botanicals International - Long Beach. CA

ProVex Plus™ contains the following ingredients per 125 milligramcapsule:

Ingredient % Formulation Amount Grape Seed Extract  20%  25 mg GinkgoBiloba Extract  8%  10 mg Bilberry Extract  8%  10 mg Grape Skin Extract 24%  30 mg Citrus Extract  40%  50 mg Total 100% 125 mg

Experiments in the Folts model using a supplement containing all of theingredients of PROVEXCV™ except the enzyme blend have shown that such asupplement is effective for eliminating CFR's at a dosage of about 20mg/Kg or less. It has been discovered that the dosage needed toeliminate CFR's observed in the Folts model can be further reduced bycombining a dietary flavonoid with an enzyme. The addition of an enzymeblend containing equal parts of two fungal proteases, an acid stableprotease and bromelain to a supplement containing all of the ingredientsof PROVEXCV™ except the enzyme blend decreased the dosage needed toeliminate CFR's in the animal model used herein from about 20 mg/Kg toabout 10 mg/Kg.

The effectiveness of PROVEXCV™ was such that the PROVEXCV™ decreased thedosage needed to eliminate CFR's in the animal model used herein fromabout 30 mg/Kg needed for ProVex Plus™ to about 10 mg/Kg for PROVEXCV™.As a result, useful doses of dietary supplements described herein areabout 30 mg/Kg or less. Preferably, an effective dosage is about 20mg/Kg or less. More preferably, an effective dosage is about 10 mg/Kg orless.

As such, the consumption of PROVEXCV™ with its antioxidant and plateletinhibitory properties may protect against the development of coronaryartery disease, acute occlusive thrombosis, death from myocardialinfarction and other conditions that are associated with plateletactivity and LDL cholesterol oxidation.

Another embodiment of the invention includes a dietary supplementdesignated PROVEXCV2™. Like ProVex Plus™ and PROVEXCV™, PROVEXCV2™ canbe used as a dietary supplement to inhibit platelet activity or LDLcholesterol oxidation. PROVEXCV2™ contains the following ingredients per1,638 milligrams:

Ingredient % Formulation Amount Enzyme Blend  4.58%   75 mg Grape SeedExtract  3.30%   54 mg Grape Skin Extract 67.77% 1,110 mg Quercetin 7.32%   120 mg Ginkgo Biloba Extract  9.71%   159 mg Bilberry Extract 7.32%   120 mg Total   100% 1,638 mg

The ingredients for the dietary supplements described herein, includingPROVEXCV2™, can be obtained from any supplier. For example, thesuppliers listed above can be used to obtain all the ingredient forPROVEXCV2™. In addition, the ingredients can be obtained from sourcesthat were not subjected to a fermentation process. For example,unfermented grape seed extract and unfermented grape skin extract can beused as ingredients for the dietary supplements described herein. Suchunfermented ingredients can be obtained from any supplier such asPolyphenolics (Canandaigua, N.Y.).

Briefly, fermentation processes are used during the production of wine.If an ingredient is obtained from a supplier involved in wineproduction, then the ingredient could be a fermented ingredient. Forexample, a fermented ingredient, such as fermented grape seed extract orfermented grape skin extract, can be any ingredient isolated from asource, such as grapes, that were subjected to fermentation. Incontrast, an unfermented ingredient can be any ingredient isolated froma source not subjected to fermentation. Such unfermented ingredients canbe more effective sources of flavonoids than fermented ingredients. Forexample, unfermented grape seed extract or unfermented grape skinextract can inhibit platelet activity or LDL cholesterol oxidation moreeffectively than fermented grape seed extract or fermented grape skinextract.

It is noted that the percentage for each ingredient in ProVex Plus™,PROVEXCV™, and PROVEXCV2™ can be changed, provided the resultingcomposition can inhibit platelet activity or LDL cholesterol oxidation.For example, the percentage of ginkgo biloba extract can be increased togreater than 10%.

In another aspect, the invention provides a method to inhibit plateletactivity or LDL cholesterol oxidation in a mammal by administering adietary supplement containing at least one flavonoid source and anenzyme that are effective for reducing platelet activity or LDLcholesterol oxidation or both.

In another aspect of the invention, a method to treat a conditionassociated with platelet activity or LDL cholesterol oxidation in amammal is provided. The method involves the step of administering adietary supplement containing at least one flavonoid source and anenzyme that are effective for reducing platelet activity or LDLcholesterol oxidation at a dosage of about 30 mg/Kg or less.

To practice the described methods, a mammal is given a dose of a dietarysupplement as described herein by an acceptable delivery method. Thedose can be administered hourly, daily, weekly or a fraction thereofdepending on the circumstances. After administration of the dietarysupplement a mammal can be evaluated for platelet activity and LDLcholesterol oxidation. The platelet activity and LDL cholesteroloxidation levels can then be compared to the same levels prior toadministering the dietary supplement.

In another aspect of the invention, an article of manufacture containinga dietary supplement that is effective for reducing platelet activityand LDL cholesterol oxidation is provided. The article is containedwithin a packaging material that is labeled to indicate that the dietarysupplement is useful for reducing platelet activity or LDL cholesteroloxidation or both in a mammal at a dosage of about 30 mg/Kg or less. Inanother embodiment of the article of manufacture, the packaging materialcan be labeled to indicate that the dietary supplement is useful totreat a condition that is associated with platelet activity or LDLcholesterol oxidation.

Any common packaging and printing method can be used to prepare thearticle of manufacture.

The invention will be further understood with reference to the followingillustrative embodiments, which are purely exemplary, and should not betaken as limiting the true scope of the present invention as describedin the claims.

EXAMPLES Example 1 Evaluating Platelet Inhibition by Dietary Supplements

Ten anesthetized dogs with coronary stenosis and medial damage wereprepared for evaluation using the methods of the Folts model asdisclosed in Folts, J., Circulation (Supplement 4), 83:3-14 (1991).Blood pressure and coronary artery blood flow were monitoredcontinuously throughout the experiment. As exemplified in FIG. 2, CFR'ssimilar to the CFR's shown in FIG. 2, which were due to acute plateletthrombus formation (APTF), occurred 8±3 times in a 30 min interval in 10dogs prior to administering PROVEXCV™. All figures showing strip chartrecordings are on the same scale, which is 10 minutes per twelvesquares. 10 mg/Kg of PROVEXCV™ was then administered by stomach tube toeach dog. As exemplified in FIG. 3, gastric administration of 10 mg/Kgof PROVEXCV™ eliminated the observed CFR's in 174±24 min in 9 of the 10dogs. The CFR's observed in the tenth dog were reduced to 2 CFR's in a30 minute interval three hours after administering 10 mg/Kg PROVEXCV™.There was no change in heart rate or arterial blood pressure produced bythe dietary flavonoids in PROVEXCV™.

When evaluated in the Folts model, platelet activity that is inhibitedby aspirin can be reactivated by administering 0.2 μg/Kg/min epinephrineintravenously. Folts, J., Circulation (Supplement 4), 83:3-14 (1991).Eight dogs that had CFR's eliminated by administering 10 mg/Kg PROVEXCV™were administered 0.2 μg/Kg/min epinephrine intravenously for 15minutes. The right side of FIG. 4 shows that CFR's eliminated by gastricadministration of 10 mg/Kg PROVEXCV™ did not reappear when epinephrinewas administered at 0.2 μg/Kg/min intravenously for 15 minutes in all 8dogs.

Ex vivo platelet activity was determined for all ten dogs using thewhole blood aggregometry method as described herein. Blood samples weretaken from each dog before and after the administration of 10 mg/Kg ofPROVEXCV™ and tested. When platelet aggregation was stimulated bycollagen, ex vivo whole blood platelet aggregation was decreased by40%±9% in all 10 dogs administered 10 mg/Kg PROVEXCV™. Unlike aspirin asshown in FIG. 5, the observed platelet aggregation increases in responseto epinephrine were not observed with PROVEXCV™.

Example 2 Evaluating LDL Cholesterol Oxidation of Dietary Supplements

As described above, LDL cholesterol oxidation is commonly measured byobserving absorbance at 234 nm (AbS_(234nm)) as a function of time whilethe LDL cholesterol is exposed to oxidative conditions. The antioxidanteffectiveness of PROVEXCV™ and other substances was determined.

LDL cholesterol was prepared from human volunteer blood samples.Isolated LDL cholesterol was then mixed with a buffer, vitamin E, ProVexPlus™ (“Orig” in FIG. 6) or PROVEXCV™ (“Curr” in FIG. 6). Experimentalsolutions of ProVex Plus™ and PROVEXCV™ were prepared at 0.5 and 1.0mg/L final concentration. The concentrations chosen for ProVex Plus™ andPROVEXCV™ were an estimate of expected blood levels of the dietarysupplements based on accepted blood absorption models. To each sample ameasured amount of copper ions was added. The amount of vitamin E usedwas comparable to the amount anticipated to be in the blood of a personadministered 400 IU of vitamin E.

After mixing, the AbS_(234nm) of each solution was then monitored as afunction of time. As shown in FIG. 6, incubating LDL cholesterol withProVex Plus™ or PROVEXCV™ at 0.5 mg/L or 1.0 mg/L protected LDLcholesterol from oxidation longer than that observed for a well knownantioxidant vitamin E at 5.3 IU/L.

As shown in FIG. 7, LDL cholesterol did not demonstrate appreciableoxidation until 45 minutes following the addition of copper ions.Vitamin E at the described concentration protected LDL cholesterol fromoxidation for about 95 minutes. 1.0 mg/L of ProVex Plus™ protected LDLcholesterol against oxidation for more than 100 minutes. Finally, 1.0mg/L of PROVEXCV™ protected LDL cholesterol against oxidation for morethan 150 minutes under these experimental conditions. As such, ProVexPlus™ and PROVEXCV™ are better antioxidants than vitamin E.

Example 3 Evaluating PROVEXCV2™

In dogs, the effects of PROVEXCV2™ on platelet activity was evaluated invivo using the Folts model and ex vivo using the whole blood plateletaggregometry test. Briefly, ten adult mongrel dogs of either sex wereanesthetized and the chest opened at the fifth intercostal space. Theleft circumflex coronary artery was dissected out and an electromagneticflow probe placed around the artery to measure coronary blood flow.Distal to the flow probe, the artery was clamped three times with aspecial surgical clamp to produce intimal and medical damage. A plasticcylinder of appropriate inside diameter was placed around the artery toproduce a 70% reduction in arterial diameter.

During the occurrence of CFR's, a blood sample was drawn for the ex vivothe whole blood platelet aggregometry test. After monitoring consistentformation of CFR's for 20 minutes, 15 mg/kg of PROVEXCV2™ wasadministered by a stomach tube. The CFR's were monitored for 3 hours.When the CFR's were abolished, a second blood sample was drawn to repeatthe ex vivo platelet aggregation test. Epinephrine (0.2 μg/kg/min) wasthen infused for 20 minutes to observe whether the CFR's could berenewed.

The ten dogs with coronary artery stenosis and intimal damage had CFR'S,due to acute platelet thrombus formation, at a frequency of 7±3 per 30minutes. After 15 mg/kg of PROVEXCV2™ was given by gastric tube, theCFR's were abolished for 164±29 minutes. The intravenous infusion ofepinephrine (0.2 μg/kg/min for 20 minutes) did not renew the CFR's inany dog. Again, epinephrine infusion after the abolishment of CFR's with5-10 mg/kg of aspirin can result in renewed CFR's in 50-60% of dogsstudied. Thus, PROVEXCV2™ appears to inhibit platelet activity morepotently than aspirin.

The activity of platelets in whole blood samples collected before andafter PROVEXCV2™ administration were evaluated ex vivo using the wholeblood platelet aggregometry test. After PROVEXCV2™ administration,platelet aggregation, induced by ADP (20 μmol/ml), decreased by 42±10%(p<0.03). In addition, the platelet aggregation produced by thecombination of epinephrine and ADP was decreased by 32±11% (p<0.05)after PROVEXCV2™ administration.

In humans, the effects of PROVEXCV2 on platelet activity also wasevaluated ex vivo using the whole blood platelet aggregometry test.Specifically, twelve healthy human volunteers (8 men, 4 women) aged22-50 years of age were recruited. They abstained from tea, alcoholicbeverages, grape products, flavonoid and vitamin supplements, and allmedications including aspirin products for one week prior to andthroughout the study. Vegetarians were excluded from the study.

On day 1 of the study for each volunteer, 18 ml of venous blood wasdrawn for the ex vivo whole blood platelet aggregometry test, between 8am and 12 noon while in the fasting state. Then, each volunteer wasinstructed to consume 5-7 capsules (about 20/mg/kg/day), depending ontheir body weight, of PROVEXCV2™ for 7-14 days. After 7-14 days, eachvolunteer returned to the laboratory in the fasting state, but havingtaken that day's dose of PROVEXCV2™, and had a second blood sampledrawn. This sample was drawn about 2-4 hours after the last dose ofPROVEXCV2™.

Briefly, 18 ml of whole blood was drawn into a syringe containing 2 mlof 3.8% sodium citrate as an anticoagulant. The blood was immediatelydiluted with an equal volume of preservative-free saline. One milliliter(1 ml) of the diluted blood was placed in a cuvette with a siliconizedstir bar and warmed to 37° C. for 5 minutes. An electrode is placed inthe cuvette to measure the impedance change, which is proportional tothe platelet aggregation. Once the baseline platelet activity wasrecorded, a high dose of collagen (12.5 μg) was added to the pre-warmedblood and placed in the aggregometer to obtain a maximum plateletaggregation response. The change in impedance produced by plateletaggregation was followed for 7 minutes. Sub-maximal doses of collagen(0.5 μg/ml), phorbol 12-myristate 13-acetate (PMA) (0.5 nmol/ml), andADP (20 μmol/ml) were used as platelet agonists. In addition, ADP (20μmol/ml) was also used as an agonist to aggregate platelets in 1 ml ofblood pre-incubated for 1 minute with epinephrine (0.5 μg/ml). Theaggregation responses were measured in duplicate in the control sampleand compared to the responses after 7-14 days of daily PROVEXCV2™.

The platelet aggregation response to collagen (0.5 μg/ml), ADP (20μmol/ml), and a phorbol ester, phorbol 12-myristate 13-acetate (PMA; 0.5nmol/ml), in the samples collected after 7-14 days of daily PROVEXCV2™administration revealed a reduction of 51.6±41.1% (p<0.005), 39.8±41.5%(p<0.005), and 17.9±10.0% (p<0.002), respectively. In addition, theplatelet aggregation in response to a combination of epinephrine (0.5μg/ml) and ADP (20 μmol/ml) in the samples collected after 7-14 days ofdaily PROVEXCV2™ administration revealed a reduction of 14.9±8.5%(p<0.05). These results suggest that the mechanism of platelet activityinhibition by PROVEXCV2™ may be through the inhibition of plateletprotein kinase C since PMA induced platelet aggregation was reducedafter 7-14 days of daily PROVEXCV2™ administration.

In addition to evaluating the effects of PROVEXCV2™ on plateletactivity, the effects of PROVEXCV2™ on LDL cholesterol oxidation wasstudied. Briefly, LDL was isolated from serum collected from ahealthy-fasting volunteer using sequential ultra-centrifugation withspins at densities of 1.006 g/ml to remove VLDL/chylomicrons and 1.063g/ml (KBr) to recover the LDL from the denser HDL and serum proteins.The duration of each spin was 3 hours using a Beckman Optima set at100,000 rpms (>4,000,000× g). The LDL was dialyzed against phosphatebuffered saline (PBS) containing EDTA for 48 hours with several changesof the buffer. The dialyzed material was then checked by electrophoresis(agarose gel) to demonstrate the absence of other lipoproteins fractionsor serum proteins. The protein concentration of the isolated LDL wasmeasured and adjusted to 0.5 g/L using EDTA-containing PBS fordilutions. The LDL solution was then aliquoted in 0.7 ml volumes intosmall glass containers with screw caps enclosures. The vials and the LDLsolutions were flushed with nitrogen to remove traces of oxygen toensure stability and stored at −80° C. until just prior to use.

LDL oxidation was performed as described above. Briefly, the LDL wasdiluted with EDTA-free PBS by mixing 100 μl of thawed LDL with 900 μl ofPBS just prior to the oxidizability study. Ten (10) μl of freshlyprepared CuCl₂ (final concentration of 5 μmol/L) was added to the LDLsolution to initiate oxidation. The rate of formation of conjugateddienes was monitored spectrophotometrically at 234 nm at 30° C. for 5hours taking absorbance readings every 3 minutes. The spectrophotometeris equipped with a six place automatic sample holder to provide amaximum of six specimens analyzed in a single run. LDL alone with PBSand CuCl₂ is used as a control when assessing the ability of PROVEXCV2™using direct mixing in vitro experiments.

The time course of LDL oxidation shows three consecutive phases: a lagphase in which there is hardly any conjugated diene formation, apropagation phase with a rapid increase in diene formation, and finallya decomposition phase. The lag phase, defined as the time period betweenthe addition of CuCl₂ and the start of propagation, is considered toreflect the LDL's susceptibility to oxidative stress.

The rate of production of conjugated dienes in a sample of LDLcholesterol is shown in FIG. 8. Native LDL cholesterol was compared toan equal sample of LDL cholesterol incubated with Vitamin E (5.3 mg/L)or with PROVEXCV2™ (1.0 mg/L). The lag time before oxidation begins wasdelayed by vitamin E and delayed to a significantly greater extent byPROVEXCV2™. This result indicates that PROVEXCV2™ is a more potentantioxidant than Vitamin E, when exposed to copper ion enhancedoxidation.

Other aspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A dietary supplement for inhibiting plateletaggregation, said supplement comprising effective amounts of a grapeseed extract, a grape skin extract, and an enzyme, wherein saidsupplement comprises 3.3 percent or more of said grape seed extract byweight, wherein said grape seed extract and said grape skin extract eachcontain one or more flavonoids, and wherein said enzyme is selected fromthe group consisting of a fungal protease, an acid stable protease, andbromelain.
 2. The supplement of claim 1, wherein said supplement is inthe form of a pill, powder, or tablet.
 3. The supplement of claim 1,wherein said supplement further comprises a bilberry extract.
 4. Thesupplement of claim 1, wherein said supplement further comprises aginkgo biloba extract.
 5. The supplement of claim 1, wherein saidsupplement further comprises quercetin.
 6. The supplement of claim 1,wherein said supplement comprises about 24 percent or less of saidenzyme by weight.
 7. The supplement of claim 1, wherein said supplementfurther comprises a ginkgo biloba extract, a bilberry extract,quercetin, a fungal protean, an acid stable protease, and bromelain. 8.The supplement of claim 7, wherein said supplement comprises about 12percent of said grape seed extract by weight, about 20 percent of saidgrape skin extract by weight, about 10 percent of said ginkgo bilobaextract by weight, about 10 percent of said bilberry extract by weight,about 24 percent of said quercetin by weight, and about 24 percent of anenzyme blend by weight.
 9. The supplement of claim 7, wherein saidsupplement comprises about 3.30 percent of said grape seed extract byweight, about 67.77 percent of said grape skin extract by weight, about9.71 percent of said ginkgo biloba extract by weight, about 7.32 percentof said bilberry extract by weight, about 7.32 percent of said quercetinby weight, and about 4.58 percent of art enzyme blend by weight.
 10. Adietary supplement for inhibiting platelet aggregation, said supplementcomprising effective amounts of a grape seed extract, a grape skinextract, and an enzyme, wherein said supplement comprises 32 percent ormore of said grape seed extract and said grape skin extract by weight,wherein said grape seed extract and said grape skin extract contain oneor more flavonoids, and wherein said enzyme is selected from the groupconsisting of a fungal protease, an acid stable protease, and bromelain.11. The supplement of claim 10, wherein said supplement is in the formof a pill, powder, or tablet.
 12. The supplement of claim 10, whereinsaid supplement further comprises a bilberry extract.
 13. The supplementof claim 10, wherein said supplement further comprises a ginkgo bilobaextract.
 14. The supplement of claim 10, wherein said supplement furthercomprises quercetin.
 15. The supplement of claim 10, wherein saidsupplement comprises about 24 percent or less of said enzyme by weight.16. The supplement of claim 10, wherein said supplement furthercomprises a ginkgo biloba extract, a bilberry extract, quercetin, afungal protease, an acid stable protease, and bromelain.
 17. Thesupplement of claim 16, wherein said supplement comprises about 12percent of said grape seed extract by weight, about 20 percent of saidgrape skit extract by weight, about 10 percent of said ginkgo bilobaextract by weight, about 10 percent of said bilberry extract by weight,about 24 percent of said quercetin by weight, and about 24 percent of anenzyme blend by weight.
 18. The supplement of claim 16, wherein saidsupplement comprises about 3.30 percent of said grape seed extract byweight, about 67.77 percent of said grape skin extract by weight, about9.71 percent of said ginkgo biloba extract by weight, about 7.32 percentof said bilberry extract by weight, about 7.32 percent of said quercetinby weight, and about 4.58 percent of an enzyme blend by weight.
 19. Amethod for inhibiting platelet aggregation in a mammal in need thereof,wherein said method comprises administering a supplement comprisingeffective amounts of a grape seed extract, a grape skin extract, and anenzyme, wherein said grape seed extract and said grape skin extractcontain one or more flavonoids, wherein said enzyme is selected from thegroup consisting of a fungal protease, an acid stable protease, andbromelain, and wherein: (a) said supplement comprises 3.3 percent ormore of said grape seed extract by weight, or (b) said supplementcomprises 32 percent or more of said grape seed extract and said grapeskin extract by weight.
 20. The method of claim 19, wherein saidsupplement is in the form of a pill, powder, or tablet.
 21. The methodof claim 19, wherein said supplement further comprises a fungalprotease, an acid stable protease, and bromelain.
 22. The method ofclaim 19, wherein said supplement further comprises a bilberry extract.23. The method of claim 19, wherein said supplement further comprises aginkgo biloba extract.
 24. The method of claim 19, wherein saidsupplement further comprises quercetin.
 25. The method of claim 19,wherein said supplement comprises about 24 percent or less of saidenzyme by weight.
 26. The method of claim 19, wherein said supplementfurther comprises a ginkgo biloba extract, a bilberry extract,quercetin, a fungal protease, an acid stable protease, and bromelain.27. The supplement of claim 26, wherein said supplement comprises about12 percent of said grape seed extract by weight, about 20 percent ofsaid grape skin extract by weight, about 10 percent of said ginkgobiloba extract by weight, about 10 percent of said bilberry extract byweight, about 24 percent of said quercetin by weight and about 24percent of an enzyme blend by weight.
 28. The supplement of claim 26,wherein said supplement comprises about 3.30 percent of said grape seedextract by weight, about 67.77 percent of sail grape skin extract byweight, about 9.71 percent of said ginkgo biloba extract by weight,about 7.32 percent of said bilberry extract by weight, about 7.32percent of said quercetin by weight, and about 4.58 percent of an enzymeblend by weight.